Overall objectives are defining proliferation and differentiation of the lymphoid precursors with the most long term repopulating ability, primitive stem cells (PSC). All lymphoid and myeloid cells are continuously descended from PSC. This project focuses on precursors that repopulate the lymphoid system over many months. Their functions are vital to health. Specific aims are: (1) To determine whether PSC are a distinct class of cells with maximal long term repopulating abilities by competitive repopulation of untreated B6 cells mixed with B6-HbbdGpi-1A cells individually marked by retroviral insertion in collaboration with I.R. Lemischka, limiting dilution in recipients with differing repopulating abilities, and testing whether enrichment separates precursors that only repopulate during a few weeks. (2) To compare effects of replication and migration on transplanted cells, by recovery after irradiation with differing portions of marrow shielded. (3) To eliminate damage from irradiation and optimize stimuli for PSC proliferation, using double W- anemic, scid mutants for repopulation without irradiation, and recipients with differing degrees of stimuli to differentiate. (4) To define early repopulation after engraftment by multipotential and lymphoid specific precursors, testing whether T and B cells, and also myeloid cells, are descended from the same precursor. (5) To identify PSC markers, enrich PSC, and develop PSC-specific monoclonal antibodies, identifying markers by effects on repopulation, and using enriched PSC to produce PSC specific monoclonals in hamsters and in W-anemic mice. Binding cells with known PSC markers that give at least 1500 fold enrichment will be used as a convenient screen. Existing monoclonals will also be tested. PSC populations will be analyzed using new techniques that directly measure repopulating and differentiation ability in vivo over many months, and estimate numbers of the precursors from which most differentiated cells are descended. These measures will be supplemented by unique retroviral insertions and limiting dilution. The former will define duration and extent of function of individual clones of precursors.